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From 2011.igem.org

8-1

After a whole night’s working, everybody goes back to sleep. Four things to do in the afternoon: 1. Extract plasmid from No.11-14 part and get their concentration. 2. Later at 17:00, extract plasmid from No.5, 4, 6, 2 part. 3. Clone the part GFP and inoculate them into LB liquid media.

8-2

Today we extract plasmid from part No. 21 and 22, measure the concentration of No. 2, 4, 5, 6 and compound 100mL of Sodium acetate. All the participants are divided into three groups: one in charge of concentrate the plasmid and connect the circuit of Group One; Team Two responsible for researching the usage of PCR in linkage and connect the circuit of Group Two; Team Three caring about how to create the deficient and gene targeting.

8-3

Today, everybody did that they should do. Team Two concentrated the plasmid extracted form Group 2, 4, 5, 6 and measures their concentration. However, they failed to find the concentrator which is much more efficient and convenient.

In the morning, we asked the teacher about the reason of the failure. The teacher believed that it was because the two restriction sites are too near to be cut once. It is better to cut them in two operations.

8-4

In the morning, we did double enzyme digestion to order 2 and 5 and single enzyme digestion to order 4 and 6，the systems of which were 50μl. We examine them after 3 hours. Double enzyme digestion in this experiment was not good while the single enzyme digestion worked well. We recycled the segments using the kit and did the second single enzyme digestion.

In the evening we recycled the 4 parts using gel electrophoresis. Two pieces of gel were used. Later we did concentration and linked them in 16℃ through the night.

8-5

We transferred and cultured the two groups that we linked yesterday. Back to the design of the experiment of Circuit Two , what we need to link are part 18, 3, 11, 16. We measure the concentration of the four parts. Nonetheless, part 18 had an extreme low concentration. So we decided to extract the plasmid again.

In the afternoon, we compounded 200 + 400 mL LB liquid media and 100mL solid LB media and sent them to sterilization.

In the evening, Group 15 and 18 were Inoculated into media. It was expected that in the next morning we would put the transformed 5α into the LB liquid media.

8-6

Another night without sleeping, at 0:00, we preserved the transgenic cells and extracted plasmid for 4, 5 and 2, 6. Then we did enzyme digestion to 3, 18, 11, and 16. Besides, part 14 and 19 were being digested at the same time. After that, we used gel electrophoresis to verify them after which a second enzyme digestion was done. And we did recycling, measuring, linking. It was quite slow.

8-7

At 7:35 in the morning, another enzyme digestion was done. Later at 8:25, we did enzyme digestion to the linked 4, 5 and 2, 6 (In order to be verified).

At 12:00, we took out 3, 11, 16, 18, 14, and 19 and did the electrophoresis and recycled them. In 11, we did not find what we needed so we could not recycle anything from that.

At 16:30, the process of linkage began and we prepared to let them stay for the whole night.